Rapamycin is a macrolide antibiotic produced by Streptomyces hygroscopicus which binds to a FK506-binding protein, FKBP, with high affinity to form a rapamycin:FKBP complex. Reported Kd values for that interaction are as low as 200 pM. The rapamycin:FKBP complex binds with high affinity to the large cellular protein, FRAP, to form a tripartite, [FKBP:rapamycin]:[FRAP], complex. In that complex rapamycin acts as a dimerizer or adapter to join FKBP to FRAP. 
A number of naturally occurring FK506 binding proteins (FKBPs) are known. See e.g. Kay, 1996, Biochem. J. 314:361-385 (review). FKBP-derived domains have been incorporated in the design of chimeric proteins for use in biological switches in genetically engineered cells. Such switches rely upon ligand-mediated multimerization of the protein components to trigger a desired biological event. See e.g. Spencer et al, 1993, Science 262:1019-1024 and PCT/US94/01617. While the potent immunosuppressive activity of FK506 would limit its utility as a multimerizing agent, especially in animals, dimers of FK506 (and related compounds) can be made which lack such immunosuppressive activity. Such dimers have been shown to be effective for multimerizing chimeric proteins containing FKBP-derived ligand binding domains.
Rapamycin, like FK506, is also capable of multimerizing appropriately designed chimeric proteins. We have previously designed biological switches using rapamycin and various derivatives or analogs thereof (xe2x80x9crapalogsxe2x80x9d) as multimerizing agents (see WO96/41865). In the case of rapamycin itself, its significant biological activities, including potent immunosuppressive activity, rather severely limit its use in biological switches in certain applications, especially those in animals or animal cells which are sensitive to rapamycin. Improved rapalogs for such applications, especially rapalogs with reduced immunosuppressive activity, would be very desirable.
A large number of structural variants of rapamycin have been reported, typically arising as alternative fermentation products or from synthetic efforts to improve the compound""s therapeutic index as an immunosuppressive agent. For example, the extensive literature on analogs, homologs, derivatives and other compounds related structurally to rapamycin (xe2x80x9crapalogsxe2x80x9d) include, among others, variants of rapamycin having one or more of the following modifications relative to rapamycin: demethylation, elimination or replacement of the methoxy at C7, C42 and/or C29; elimination, derivatization or replacement of the hydroxy at C13, C43 and/or C28; reduction, elimination or derivatization of the ketone at C14, C24 and/or C30; replacement of the 6-membered pipecolate ring with a 5-membered prolyl ring; and alternative substitution on the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl ring. Additional historical information is presented in the background sections of U.S. Pat. Nos. 5,525,610; 5,310,903 and 5,362,718.
U.S. Pat. No. 5,527,907 is illustrative of the patent literature. That document discloses a series of compounds which were synthesized in an effort to make immunosuppressive rapalogs with reduced side effects. The compounds are disclosed via seven generic structural formulas, each followed by extensive lists (two to five or more columns of text each) setting forth possible substituents at various positions on the rapamycin ring. The document includes over 180 synthetic examples. The many structural variants of that invention were reported to be potent immunosuppressive agents.
This invention provides methods and materials for multimerizing chimeric proteins in genetically engineered cells using improved rapalogs, preferably while avoiding the immunosuppressive effects of rapamycin.
The genetically engineered cells contain one or more recombinant nucleic acid constructs encoding specialized chimeric proteins as described herein. Typically a first chimeric protein contains one or more FKBP domains which are capable of binding to an improved rapalog of this invention. This first chimeric protein is also referred to herein as an xe2x80x9cFKBP fusion proteinxe2x80x9d and further comprises at least one protein domain heterologous to at least one of its FKBP domains. The complex formed by the binding of the FKBP fusion protein to the rapalog is capable of binding to a second chimeric protein which contains one or more FRB domains (the xe2x80x9cFRB fusion proteinxe2x80x9d). The FRB fusion protein further comprises at least one protein domain heterologous to at least one of its FRB domains. In some embodiments, the FKBP fusion protein and the FRB fusion protein are different from one another. In other embodiments, however, the FKBP fusion protein is also an FRB fusion protein. In those embodiments, the chimeric protein comprises one or more FKBP domains as well as one or more FRB domains. In such cases, the first and second chimeric proteins may be the same protein, may be referred to as FKBP-FRB fusion proteins and contain at least one domain heterologous to the FKBP and/or FRB domains.
The chimeric proteins may be readily designed, based on incorporation of appropriately chosen heterologous domains, such that their multimerization triggers one-or more of a wide variety of desired biological responses. The nature -of the biological response triggered by rapalog-mediated complexation is determined by the choice of heterologous domains in the fusion proteins. The heterologous domains are therefore referred to as xe2x80x9cactionxe2x80x9d or xe2x80x9ceffectorxe2x80x9d domains. The genetically engineered cells for use in practicing this invention will contain one or more recombinant nucleic acid constructs encoding the chimeric proteins, and in certain applications, will further contain one or more accessory nucleic acid constructs, such as one or more target gene constructs. Illustrative biological responses, applications of the system and types of accessory nucleic acid constructs are discussed in detail below.
A system involving related materials and methods is disclosed in WO 96/41865 (Clackson et al) and is expected to be useful in a variety of applications including, among others, research uses and therapeutic applications. That system involves the use of a multimerizing agent comprising rapamycin or a rapalog of the generic formula: 
wherein U is xe2x80x94H, xe2x80x94OR1, xe2x80x94SR1, xe2x80x94OC(O)R1, xe2x80x94OC(O)NHR1, xe2x80x94NHR1, xe2x80x94NHC(O)R1, xe2x80x94NHSO2xe2x80x94R1 or xe2x80x94R2; R2 is a substituted aryl or allyl or alkylaryl (e.g. benzyl or substituted benzyl); V is xe2x80x94R3 or (xe2x95x90O); W is xe2x95x90O, xe2x95x90NR4xe2x95x90NOR4, xe2x95x90NNHR4, xe2x80x94NHOR4, xe2x80x94NHNHR4, xe2x80x94OR4, xe2x80x94OC(O)R4, xe2x80x94OC(O)NR4 or xe2x80x94H; Y is xe2x80x94OR5, xe2x80x94OC(O)R5 or xe2x80x94OC(O)NHR5; Z is xe2x95x90O, xe2x80x94OR6, xe2x80x94NR6, xe2x80x94H, xe2x80x94NC(O)R6, xe2x80x94OC(O)R6 or xe2x80x94OC(O)NR6; R3 is H, xe2x80x94R7, xe2x80x94C(O)R7, xe2x80x94C(O)NHR7 or C-28/C-30 cyclic carbonate; and R4 is H or alkyl; where R1, R4, R5, R6 and R7 are independently selected from H, alkyl, alkylaryl or aryl, as those terms are defined in WO 96/41865. A number of rapalogs are specifically disclosed in that document.
The subject invention is based upon a system similar to that disclosed in WO 96/41865, but involves the use of improved rapalogs as the multimerizing agents. The subject invention thus provides a method for multimerizing chimeric proteins in cells which comprises (a) providing appropriately engineered cells containing nucleic acid constructs for directing the expression of the desired chimeric protein(s) and any desired accessory recombinant constructs, and (b) contacting the cells with an improved rapalog or a pharmaceutically acceptable derivative thereof as described herein. The rapalog forms a complex containing itself and at least two molecules of the chimeric protein(s). Improved rapalogs for use in this invention include the following.
One class of improved rapalogs for use in this invention consists of those compounds which comprise the substructure shown in Formula I: 
bearing any number of a variety of substituents, and optionally unsaturated at one or more carbonxe2x80x94carbon bonds unless specified to the contrary herein, which have a substantially reduced immunosuppressive effect as compared with rapamycin. By a xe2x80x9csubstantially reduced immunosuppressive effectxe2x80x9d we mean that the rapalog has less than 0.1, preferably less than 0.01, and even more preferably, less than 0.005 times the immunosuppressive effect observed or expected with an equimolar amount of rapamycin, as measured either clinically or in an appropriate in vitro or in vivo surrogate of human immunosuppressive activity, preferably carried out on tissues of lymphoid origin, or alternatively, that the rapalog yields an EC50 value in such an in vitro assay which is at least ten times, preferably at least 100 times and more preferably at least 250 times larger than the EC50 value observed for rapamycin in the same assay.
One appropriate in vitro surrogate of immunosuppression in a human patient is inhibition of human T cell proliferation in vitro. This is a conventional assay approach that may be conducted in a number of well known variations using various human T cells or cells lines, including among others human PBLs and Jurkat cells. A rapalog may thus be assayed for human immunosuppressive activity and compared with rapamycin. A decrease in immunosuppressive activity relative to rapamycin measured in an appropriate in vitro assay is predictive of a decrease in immunosuppressive activity in humans, relative to rapamycin. Such in vitro assays may be used to evaluate the rapalog""s relative immunosuppressive activity.
A variety of illustrative examples of such rapalogs are disclosed herein. This class of improved rapalogs includes, among others, those which bind to human FKBP12, or inhibit its rotamase activity, within an order of magnitude of results obtained with rapamycin in any conventional FKBP binding or rotamase assay.
Other classes of improved rapalogs for use in this invention are defined with reference to the structure shown in Formula II: 
and,
one of RC7a and RC7b is H and the other is xe2x80x94H, halo, xe2x80x94R2, xe2x80x94OR1, xe2x80x94SR1, xe2x80x94OC(O)R1 or xe2x80x94OC(O)NHR1, xe2x80x94NHR1, xe2x80x94NR1R2, xe2x80x94NHC(O)R1, or xe2x80x94NHxe2x80x94SO2xe2x80x94R1 where R2=aliphatic, heteroaliphatic, aryl, heteroaryl or alkylaryl (e.g. benzyl or substituted benzyl);
RC30 is halo, xe2x80x94OR3 or (xe2x95x90O);
RC24 is xe2x95x90O, xe2x95x90NR4, xe2x95x90NOR4, xe2x95x90NNHR4, xe2x95x90NHOR4, xe2x95x90NHNHR4, xe2x95x90OR4, xe2x80x94OC(O)R4or xe2x80x94OC(O)NR4, halo or xe2x80x94H;
RC13 and RC28 are independently H, halo, xe2x80x94OR3, xe2x80x94OR5, xe2x80x94OC(O)R5, xe2x80x94OC(O)NHR5, xe2x80x94SR5, xe2x80x94SC(O)R5, xe2x80x94SC(O)NHR5, xe2x80x94NR5R5xe2x80x2 or xe2x80x94N(R5)(CO)R5xe2x80x2;
RC14 is xe2x95x90O, xe2x80x94OR6, xe2x80x94NR6, xe2x80x94H, xe2x80x94NC(O)R6, xe2x80x94OC(O)R6or xe2x80x94OC(O)NR6;
R3 is H, xe2x80x94R7, xe2x80x94C(O)R7 or xe2x80x94C(O)NHR7 or a cyclic moiety (e.g., carbonate) bridging C28 and C30; and,
RC29 is H or OR11 (e.g., OH or OMe);
where each substituent may be present in either stereochemical orientation unless otherwise indicated, and where wach occurence of R1, R4, R5, R6, R7, R9, R10 and R11 is independently selected from H, aliphatic, heteroaliphatic, aryl, and heteroaryl; and R8 is H, halo, xe2x80x94CN, xe2x95x90O, xe2x80x94OH, xe2x80x94NR9R10, OSO2CF3, OSO2F, OSO2R4xe2x80x2, OCOR4xe2x80x2, OCONR4xe2x80x2R5xe2x80x2, or OCON(OR4xe2x80x2)R5xe2x80x2.
Improved rapalogs useful in praticing this invention, including rapalogs of Formula II, may contain substituents in any of the possible stereoisomeric orientations, and may comprise one stereoisomer subtantially free of other stereoisomers ( greater than 90%, and preferable  greater than 95%, free from other stereoisomers on a molar basis) or may comprise a mixture of stereoisomers.
One class of improved rapalogs for use in this invention which are of particular interest are rapalogs of Formula II wherein one or both of RC13 and RC28 is independently H, halo, xe2x80x94OR3, xe2x80x94OR5, xe2x80x94OC(O)R5, xe2x80x94OC(O)NHR5, xe2x80x94SR5, xe2x80x94SC(O)R5, xe2x80x94SC(O)NHR5, xe2x80x94NR5R5xe2x80x2 or xe2x80x94N(R5)(CO)R5xe2x80x2, where each halo moiety is independently selected form F, Cl, Br and I. One subset of such compounds differs in structure from rapamycin only at one or both of RC13 and RC28. Another subset of such compounds differs in structure from rapamycin at one or more additional positions, as set forth above in connection with Formula II or in connection with any of the other classes of improved rapalogs noted herein. Compounds of both subsets which are of particular note are those in which one or both of RC13 and RC28 is a helo substituent, independently selected from F, Cl, Br and I, or a substituted or unsubstituted amino moiety or acylated derivative thereof. These compounds include the 13-halo rapamycins, 28-halo rapamycins, 13, 28-dihalo rapamycins and related compounds in which one or more other moities (e.g. one or both substituents at C7, for instance), in addition to the C13 and C28 substituents, differ from the corresponding moiety(ies) in rapamycin.
Another class of improved rapalogs for use in this invention which are of particular interest are rapalogs of Formula II wherein both RC24 and RC30 are other than xe2x95x90O. This class includes 24, 30-tetrahydro rapamycin and mono and diethers thereof and the 24,30-dihalo rapamycins. One subset of such compounds differs in structure from rapamycin only at RC24 and RC30. Another subset of such compounds differs in structure from rapamycin at one or more additional positions (e.g. one or both substituents at C7, for instance), as set forth above in connection with Formula II or in connection with any of the other classes of improved rapalogs noted herein.
Another class of improved rapalogs for use in this invention which are of particular interest are rapalogs of Formula II wherein RC7a and RC7b are moieties other than a substituted or unsubstituted allyl group or a methoxy moiety. This class includes rapalogs in which one of RC7a and RC7b is H and the other is phenyl, di- or tri-substituted phenyl or a mono- or di-substituted heterocyclic moiety. Illustrative examples include among others, o,p-dialkoxyphenyl substituents (e.g., o,p-dimethoxyphenyl, o-methoxy-p-ethoxyphenyl, o-ethoxy-p-methoxyphenyl, o,p-diethoxyphenyl, o,p-di (n- or iso-)propoxyphenyl, etc.), trialkoxyphenyl substituents, monosubstituted heterocycles such as methylthiophene, etc. One subset of such compounds differs in structure from rapamycin only at RC24 and RC30. Another subset of such compounds differs in structure from rapamycin at one or more additional position, as set forth above in connection with Formula II or in connection with any of the other classes of improved rapalogs noted herein.
Another class of improved rapalogs for use in this invention which are of particular interest are rapalogs of Formula II wherein n is 1. This class of rapalogs includes rapalogs comprising a prolyl ring system in place of a pipicolate ring system. One subset of such compounds differs in structure from rapamycin only with respect to the pipicolate ring system. Another subset of such compounds differs in structure from rapamycin with respect to one or more additional structural features (e.g. one or both substituents at C7, for instance), as set forth above in connection with Formula II or in connection with any of the other classes of improved rapalogs noted herein.
Another class of improved rapalogs for use in this invention which are of particular interest are rapalogs of Formula II wherein moiety xe2x80x9caxe2x80x9d is other than 
One subset of such compounds differs in structure from rapamycin only with respect to the ring system, xe2x80x9caxe2x80x9d. Another subset of such compounds differs in structure from rapamycin with respect to one or more additional structural features (e.g. one or both substituents at C7, for instance), as set forth above in connection with Formula II or in connection with any of the other classes of improved rapalogs noted herein. This class of rapalogs include the class of 43-epi-rapalogs in which the hydroxyl moiety at position 43 has the opposite stereochemical orientation with that shown immediately above, is a mixture of stereoisomers of the 43-hydroxyl group or contains derivatives of any of the foregoing, including ethers, esters, carbamates, halides and other derivatives of any of the foregoing position 43 rapalogs. This class further includes rapalogs in which the cyclohexyl ring is otherwise substituted and/or contains 5 ring atoms in place of the characteristic substituted cyclohexyl ring of rapamycin.
Again, the improved rapalogs as described herein are used in a method for multimerizing chimeric proteins in genetically engineered cells. The method involves (a) providing appropriately engineered cells containing nucleic acid constructs for directing the expression of the desired chimeric proteins (and any desired accessory recombinant constructs), and (b) contacting the cells with an improved rapalog or a pharmaceutically acceptable derivative thereof.
In one embodiment, at least one of the chimeric proteins contains at least one FKBP domain whose peptide sequence differs from a naturally occurring FKBP peptide sequence, e.g. the peptide sequence of human FKBP12, at up to ten amino acid residues in the peptide sequence. Preferably the number of changes in peptide sequence is limited to five, and more preferably to 1, 2, or 3. In embodiments in which the rapalog comprises a structural modification relative to rapamycin at RC28, at RC24 and RC30, and/or at RC7a and/or RC7b, it is also of special interest that at least one of the chimeric proteins contains at least one FKBP domain comprising at least one amino acid replacement relative to the sequence of a naturally occurring FKBP, especially a mammalian FKBP such as human FKBP12. Mutations of particular interest include replacement of either or both of Phe36 and Phe99 of human FKBP12 sequence with independently selected replacement amino acids, e.g. valine, methionine, alanine or serine.
In another embodiment, at least one of the chimeric proteins contains at least one FRB domain whose peptide sequence differs from a naturally occurring FRB peptide sequence, e.g. the FRB domain of human FRAP, at up to ten amino acid residues in the peptide sequence. Preferably the number of changes in peptide sequence is limited to five, and more preferably to 1, 2, or 3. in many cases it will be preferred that the FRB domain contains a single amino acid replacement relative to the peptide sequence of the corresponding FRB domain of human FRAP or some other mammalian FRAP/TOR species. Mutations of particular interest include replacement of one or more of T2098, D2102, Y2038, F2039, K2095 of an FRB domain derived from human FRAP with independently selected replacement amino acids, e.g. A, N, H, L, or S. Also of interest are the replacement of one or more of F1975, F1976, D2039 and N2035 of an FRB domain derived from yeast TOR1, or the replacement of one or more of F1978, F1979, D2042 and N2038 of an FRB domain derived from yeast TOR2, with independently selected replacement amino acids, e.g. H, L, S, A or V.
In certain embodiments the chimeric protein(s) contain at least one modification in peptide sequence, preferably up to three modifications, relative to naturally occurring sequences, in both one or more FKBP domains and one or more FRB domains.
As mentioned previously, in the various embodiments of this invention, the chimeric protein(s) contain one or more xe2x80x9cactionxe2x80x9d or xe2x80x9ceffectorxe2x80x9d domains which are heterologous with respect to the FKBP and/or FRB domains. Effector domains may be selected from a wide variety of protein domains including DNA binding domains, transcription activation domains, cellular localization domains and signaling domains (i.e., domains which are capable upon clustering or multimerization, of triggering cell growth, proliferation, differentiation, apoptosis, gene transcription, etc.). A variety of illustrative effector domains which may be used in practising this invention are disclosed in the various scientific and patent documents cited herein.
For example, in certain embodiments, one fusion protein contains at least one DNA binding domain (e.g., a GAL4 or ZFHD1 DNA-binding domain) and another fusion protein contains at least one transcription activation domain (e.g., a VP16 or p65 transcription activation domain). Ligand-mediated association of the fusion proteins represents the formation of a transcription factor complex and leads to initiation of transcription of a target gene linked to a DNA sequence recognized by (i.e., capable of binding with) the DNA-binding domain on one of the fusion proteins.
In other embodiments, one fusion protein contains at least one domain capable of directing the fusion protein to a particular cellular location such as the cell membrane, nucleus, ER or other organelle or cellular component. Localization domains which target the cell membrane, for example, include domains such as a myristoylation site or a transmembrane region of a receptor protein or other membrane-spanning protein. Another fusion protein can contain a signaling domain capable, upon membrane localization and/or clustering, of activating a cellular signal transduction pathway. Examples of signaling domains include an intracellular domain of a growth factor or cytokine receptor, an apoptosis triggering domain such as the intracellular domain of FAS or TNF-R1, and domains derived from other intracellular signaling proteins such as SOS, Raf, Ick, ZAP-70, etc. A number of signaling proteins are disclosed in PCT/US94/01617 (see e.g. pages 23-26). In still other embodiments, each of the fusion proteins contains at least one FRB domain and at least one FKBP domain, as well as one or more heterologous domains. Such fusion proteins are capable of homodimerization and triggering signaling in the presence of the rapalog. In general, domains containing peptide sequence endogenous to the host cell are preferred in applications involving whole organisms. Thus, for human gene therapy applications, domains of human origin are of particular interest.
Recombinant nucleic acid constructs encoding the fusion proteins are also provided, as are nucleic acid constructs capable of directing their expression, and vectors containing such constructs for introducing them into cells, particularly eukaryotic cells, of which yeast and animal cells are of particular interest. In view of the constituent components of the fusion proteins, the recombinant DNA molecules which encode them are capable of selectively hybridizing (a) to a DNA molecule encoding a polypeptide comprising an FRB domain or FKBP domain and (b) to a DNA molecule encoding the heterologous domain or a protein from which the heterologous protein domain was derived. DNAs are also encompassed which would be capable of so hybridizing but for the degeneracy of the genetic code.
Using nucleic acid sequences encoding the fusion proteins, nucleic acid constructs for directing their expression in eukaryotic cells, and vectors or other means for introducing such constructs into cells, especially animal cells, one may genetically engineer cells, particularly animal cells, preferably mammlian cells, and most preferably human cells, for a number of important uses. To do so, one first provides an expression vector or nucleic acid construct for directing the expression in a eukaryotic (preferably animal) cell of the desired chimeric protein(s) and then introduces the recombinant DNA into the cells in a manner permitting DNA uptake and expression of the introduced DNA in at least a portion of the cells. One may use any of the various methods and materials for introducing DNA into cells for heterologous gene expression, a variety of which are well known and/or commercially available.
One object of this invention is thus a method for multimerizing fusion proteins, such as described herein, in cells, preferably animal cells. To recap, one of the fusion proteins is capable of binding to the improved rapalog of this invention and contains at least one FKBP domain and at least one domain heterologous thereto. The second fusion protein contains at least one FRB domain and at least one domain heterologous thereto and is capable of forming a tripartite complex with the first fusion protein and one or more molecules of the improved rapalog. In some embodiments one or more of the heterologous domains present on one of the fusion proteins are also present on the other fusion protein, i.e., the two fusion proteins have one or more common heterologous domains. In other embodiments, each fusion protein contains one or more different heterologous domains.
The method comprises contacting appropriately engineered cells with the improved rapalog by adding the rapalog to the culture medium in which the cells are located or administering the rapalog to the organism in which the cells are located. The cells are preferably eukaryotic cells, more preferably animal cells, and most preferably mammalian cells. Primate cells, especially human cells, are of particular interest. Administration of the improved rapalog to a human or non-human animal may be effected using any pharmaceutically acceptable formulation and route of administration. Oral administration of a pharmaceutically acceptable composition containing the improved rapalog together with one or more pharmaceuticaly acceptable carriers, buffers or other excipients is currently of greatest interest.
A specific object of this invention is a method, as otherwise described above, for inducing transcription of a target gene in a rapalog-dependent manner. The cells typically contain, in addition to recombinant DNAs encoding the two fusion proteins, a target gene construct which comprises a target gene operably linked to a DNA sequence which is responsive to the presence of a complex of the fusion proteins with rapamycin or a rapalog. The target gene construct may be recombinant, and the target gene and/or a regulatory nucleic acid sequence linked thereto may be heterologous with respect to the host cell. In certain embodiments the cells are responsive to contact with an improved rapalog which binds to the FKBP fusion protein and participates in a complex with a FRB fusion protein with a detectable preference over binding to endogenous FKBP and/or FRB-containing proteins of the host cell.
Another specific object of this invention is a method, as otherwise described above, for inducing cell death in a rapalog-dependent manner. In such cells, at least one of the heterologous domains on at least one fusion protein, and usually two fusion proteins, is a domain such as the intracellular domain of FAS or TNF-R1, which, upon clustering, triggers apoptosis of the cell.
Another specific object of this invention is a method, as otherwise described above, for inducing cell growth, differentiation or proliferation in a rapalog-dependent manner. In such cells, at least one of the heterologous domains of at least one of the fusion proteins is a signaling domain such as, for example, the intracellular domain of a receptor for a hormone which mediates cell growth, differentiation or proliferation, or a downstream mediator of such a receptor. Cell growth, differentiation and/or proliferation follows clustering of such signalling domains. Such clustering occurs in nature following hormone binding, and in engineered cells of this invention following contact with an improved rapalog.
Cells of human origin are preferred for human gene therapy applications, although cell types of various origins (human or other species) may be used, and may, if desired, be encapsulated within a biocompatible material for use in human subjects.
Also provided are materials and methods for producing the foregoing engineered cells. This object is met by providing recombinant nucleic acids, typically DNA molecules, encoding the fusion proteins, together with any desired ancillary recombinant nucleic acids such as a target gene construct, and introducing the recombinant nucleic acids into the host cells under conditions permitting nucleic acid uptake by cells. Such transfection may be effected ex vivo, using host cells maintained in culture. Cells that are engineered in culture may subsequently be introduced into a host organism, e.g. in ex vivo gene therapy applications. Doing so thus constitutes a method for providing a host organism, preferably a human or non-human mammal, which is responsive (as described herein) to the presence of an improved rapalog as provided herein. Alternatively transfection may be effected in vivo, using host cells present in a human or non-human host organism. In such cases, the nucleic acid molecules are introduced directly into the host organism under conditions permitting uptake of nucleic acids by one or more of the host organism""s cells. This approach thus constitutes an alternative method for providing a host organism, preferably a human or non-human mammal, which is responsive (as described herein) to the presence of an improved rapalog. Various materials and methods for the introduction of DNA and RNA into cells in culture or in whole organisms are known in the art and may be adapted for use in practicing this invention.
Other objects are achieved using the engineered cells described herein. For instance, a method is provided for multimerizing fusion proteins of this invention by contacting cells engineered as described herein with an effective amount of the improved rapalog permitting the rapalog to form a complex with the fusion proteins. In embodiments in which multimerization of the fusion proteins triggers transcription of a target gene, this constitutes a method for activating the expression of the target gene. In embodiments in which the fusion proteins contain one or more signaling domains, this constitutes a method for activating a cellular signal transduction pathway. In specific embodiments in which the signaling domains are selected based on their ability following clustering to trigger cell growth, proliferation, diffeentiation or cell death, improved rapalog-mediated clustering constitutes a method for actuating cell growth, proliferation, diffeentiation or cell death, as the case may be. These methods may be carried out in cell culture or in whole organisms, including human patients. In the former case, the rapamycin or rapalog is added to the culture medium. In the latter case, the rapamycin or rapalog (which may be in the form of a pharmaceutical or veterinary composition) is administered to the whole organism, e.g., orally, parenterally, etc. Preferably, the dose of the improved rapalog administered to an animal is below the dosage level that would cause undue immunosuppression in the recipient.
Also disclosed are kits for use in the genetic engineering of cells or human or non-human animals as described herein. One such kit contains one or more recombinant nucleic acid constructs encoding fusion proteins of this invention. The recombinant nucleic acid constructs will generally be in the form of eukaryotic expression vectors suitable for introduction into animal cells and capable of directing the expression of the fusion proteins therein. Such vectors may be viral vectors as described elsewhere herein. The kit may also contain a sample of an improved rapalog of this invention capable of forming a complex with the encoded fusion proteins. The kit may further contain a multimerization antagonist such as FK506 or some other compound capable of binding to one of the fusion proteins but incapable of forming a complex with both. In certain embodiments, the recombinant nucleic acid constructs encoding the fusion proteins will contain a cloning site in place of DNA encoding one or more of the heterologous domains, thus permitting the practitioner to introduce DNA encoding a heterologous domain of choice. In some embodiments the kit may also contain a target gene construct containing a target gene or cloning site linked to a DNA sequence responsive to the presence of the complexed fusion proteins, as described in more detail elsewhere. The kit may contain a package insert identifying the enclosed nucleic acid construct(s), and/or instructions for introducing the construct(s) into host cells or organisms.